In most cell types, microtubules are organized by the centrosome, an organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome "matures" or gradually acquires increased levels of microtubule nucleating capacity and PCM. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. Specifically, our goals are to identify the factors that regulate these processes and to understand how they function on a molecular level. [unreadable] [unreadable] During 2008, we continued to study two factors that regulate centrosome duplication and have initiated studies on two others. All were identified in a screen for genes that genetically interact with zyg-1, a conserved upstream regulator of centrosome duplication. SZY-20 is a negative regulator of ZYG-1 and localizes to centrosomes. We have demonstrated that SZY-20 limits centrosome size and that in its absence centrosomes are enlarged. These centrosomes possess elevated levels of ZYG-1 and PCM components such as SPD-2, SPD-5, and gamma-tubulin. Further, we have found that these enlarged centrosomes nucleate more microtubules than their wild-type counterparts and that the enlarged centrosome phenotype is associated with defects in microtubule-dependent processes. Of particular significance we find that two centrosome duplication factors, ZYG-1 and SAS-6 are involved in defining centrosome size. In particular, inhibition of ZYG-1 in the szy-20 mutant restores normal centrosome size and rescues the microtubule-related defects. Importantly, thin section transmission electron microscopy has revealed that szy-20 mutants possess centrioles of normal size. This and other data argue that the role of ZYG-1 in controlling centrosome size is separable from its role in centriole replication. [unreadable] [unreadable] Last year we cloned the szy-20 gene and found that it encodes a widely conserved, yet unstudied protein. At the time, our analysis indicated that SZY-20 was likely to be an RNA-binding protein. To address this we performed overexpression studies and have found that overexpressing SZY-20 reduces centrosome size in wild-type embryos yet no effect is seen when an RNA-binding defective mutant is overexpressed. Our data indicates that SZY-20 regulates the level of ZYG-1 at the centrosome and may act by controlling the translation, stability, or localization of centrosome-associated mRNAs. [unreadable] [unreadable] We are also continuing our studies on SZY-5, another negative regulator of ZYG-1. SZY-5 encodes a zinc-finger protein but does not seem to regulate centrosome-levels of ZYG-1, indicating that it interacts with ZYG-1 in a manner distinctly different from that of SZY-20. We have postulated that SZY-5 might control the expression of one or more centrosome duplication factors and have used RT-PCR and quantitative immunostaining to address this issue. So far, SZY-5 does not appear to affect the expression or localization of any of the known centrosome duplication factors. To further define the function of SZY-5 we have sought to determine its subcellular distribution and to characterize defects that arise in its absence. Toward this end, we have raised an antibody to SZY-5 and find that it stains nuclei and cytoplasmic granules. We have also found that embryos lacking SZY-5 display defects in osmotic integrity, cell cycle progression, polar body extrusion, cytokinesis, and anaphase spindle positioning. As some of the processes affected by the szy-5 mutation involve centrosome function, we are examining centrosome structure and function in the mutant. Finally, we are initiating a proteomics approach to study SZY-5, as the identification of interacting factors should help us understand the role of SZY-5 in centrosome duplication.[unreadable] [unreadable] This past year we have also initiated analysis of two other factors that interact with ZYG-1. Mutation of the szy-1 gene suppresses the centrosome duplication defect of zyg-1 mutants and disrupts normal centrosome morphology. We have mapped szy-3 to a small physical interval and hope to identify the gene using RNAi to phenocopy szy-1 mutant defects. The szy-2 gene is also another suppressor of ZYG-1. We have cloned szy-2 and find that it encodes a regulatory subunit of protein phosphatase 1 (PP1) called I-2. We find that I-2 opposes the activity of ZYG-1 and are currently testing to see if I-2 negatively or positively regulates PP1. Since ZYG-1 is a kinase, PP1 may interact directly with ZYG-1 substrates. In addition to its interaction with ZYG-1, we find that I-2 is required for proper chromosome segregation.